The present invention is directed to unnatural polypeptide-like molecules which are oligomers or polymers of constrained, di-substituted imino carboxylic acids, methods of generating combinatorial libraries using these residues, and combinatorial libraries formed thereby.
Chemists have long sought to extrapolate the power of biological catalysis and recognition to synthetic systems. These efforts have focused largely on low molecular weight catalysts and receptors. Most biological systems, however, rely almost exclusively on large polymers such as proteins and RNA to perform complex chemical functions.
Proteins and RNA are unique in their ability to adopt compact, well-ordered conformations. These two biopolymers are unique also because they can perform complex chemical operations (e.g., catalysis, highly selective recognition, etc.). Folding is linked to function in both proteins and RNA because the creation of an xe2x80x9cactive sitexe2x80x9d requires proper positioning of reactive groups. Consequently, there has been a long-felt need to identify synthetic polymer backbones which display discrete and predictable folding propensities (hereinafter referred to as xe2x80x9cfoldamersxe2x80x9d) to mimic natural biological systems. Such backbones will provide molecular xe2x80x9ctoolsxe2x80x9d to probe the functionality of large-molecule interactions (e.g. proteinxe2x80x94protein and protein-RNA interactions).
Much work on xcex2-amino acids and peptides synthesized therefrom has been performed by a group led by Dieter Seebach in Zurich, Switzerland. See, for example, Seebach et al. (1996) Helv. Chim. Acta. 79:913-941; and Seebach et al. (1996) Helv. Chim. Acta. 79:2043-2066. In the first of these two papers Seebach et al. describe the synthesis and characterization of a xcex2-hexapeptide, namely (H-xcex2-HVal-xcex2-HAla-xcex2-HLeu)2xe2x80x94OH. Interestingly, this paper specifically notes that prior art reports on the structure of xcex2-peptides have been contradictory and xe2x80x9cpartially controversial.xe2x80x9d In the second paper, Seebach et al. explore the secondary structure of the above-noted 13-hexapeptide and the effects of residue variation on the secondary structure.
Dado and Gellman (1994) J. Am. Chem. Soc. 116:1054-1062 describe intramolecular hydrogen bonding in derivatives of xcex2-alanine and xcex3-amino butyric acid. This paper postulates that xcex2-peptides will fold in manners similar to xcex1-amino acid polymers if intramolecular hydrogen bonding between nearest neighbor amide groups on the polymer backbone is not favored.
Suhara et al. (1996) Tetrahedron Lett. 37(10):1575-1578 report a polysaccharide analog of a xcex2-peptide in which D-glycocylamine derivatives are linked to each other via a C-1 xcex2-carboxylate and a C-2 xcex1-amino group. This class of compounds has been given the trivial name xe2x80x9ccarbopeptoids.xe2x80x9d
Regarding methods to generate combinatorial libraries, several recent reviews are available. See, for instance, Ellman (1996) Acc. Chem. Res. 29:132-143 and Lam et al. (1997) Chem. Rev. 97:411-448.
The present invention is drawn to a genus of oligomers and polymers of conformationally-restricted, di-substituted imino carboxylic acids. The preferred oligomers and polymers of the invention strongly favor a discrete secondary structure (although this is not a requirement of the invention). These stable secondary structures include helices analogous to the well-known poly(proline) II helical structure seen in xcex1-amino acids.
More specifically, the invention is directed to compounds comprising formula:
Xxe2x80x94{A}nxe2x80x94Y 
wherein n is an integer greater than 1; and
each A, independent of every other A, is selected from the group consisting of: 
wherein R1 and R2 are independently selected from the group consisting of hydrogen, hydroxy, linear or branched C1-C16-alkyl, alkenyl, or alkynyl; mono-or di-C1-C16 alkylamino; mono- or bicyclic aryl; mono- or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C16-alkyl; mono- or bicyclic heteroaryl-C1-C16-alkyl; xe2x80x94(CH2)0-6xe2x80x94OR3, xe2x80x94(CH2)0-6xe2x80x94SR3, xe2x80x94(CH2)0-6xe2x80x94S(xe2x95x90O)xe2x80x94CH2xe2x80x94 R3, xe2x80x94(CH2)0-6xe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94R3, xe2x80x94(CH2)0-6xe2x80x94NR3R3, xe2x80x94(CH2)0-6xe2x80x94NHC(xe2x95x90O)R3, xe2x80x94(CH2)0-6xe2x80x94NHS(xe2x95x90O)2xe2x80x94CH2xe2x80x94R3, xe2x80x94(CH2)0-6Oxe2x80x94(CH2)2-6xe2x80x94R4, xe2x80x94(CH2)0-6xe2x80x94Sxe2x80x94(CH2)2-6xe2x80x94R4, xe2x80x94(CH2)0-6xe2x80x94Sxe2x95x90O)xe2x80x94(CH2)2-6xe2x80x94R4, xe2x80x94(CH2)0-6xe2x80x94S(xe2x95x90O)2xe2x80x94(CH2)2-6xe2x80x94R4, xe2x80x94(CH2)0-6xe2x80x94NHxe2x80x94(CH2)2-6xe2x80x94R4, xe2x80x94(CH2)0-6xe2x80x94Nxe2x80x94{(CH2)2-6xe2x80x94R4}2, xe2x80x94(CH2)0-6xe2x80x94NHC(xe2x95x90O)xe2x80x94(CH2)2-6xe2x80x94R4, and xe2x80x94(CH2)0-6xe2x80x94NHS(xe2x95x90O)2xe2x80x94(CH2)2-6xe2x80x94R4; wherein
R3 is independently selected from the group consisting of hydrogen, C1-C6-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl, mono- or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, mono- or bicyclic heteroaryl-C1-C6-alkyl; and
R4 is selected from the group consisting of hydroxy, C1-C6-alkyloxy, aryloxy, heteroaryloxy, thio, C1-C6-alkylthio, C1-C6-alkylsufinyl, C1-C6-alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl, heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- or di-C1-C6-alkylamino, mono- or diarylamino, mono- or diheteroarylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-aryl-N-heteroarylamino, aryl-C1-C6-alkylamino, carboxylic acid, carboxamide, mono- or di-C1-C6-alkylcarboxamide, mono- or diarylcarboxamide, mono- or diiheteroarylcarboxamide, N-alkyl-N-arylcarboxamide, N-alkyl-N-heteroarylcarboxamide, N-aryl-N-heteroarylcarboxamide, sulfonic acid, sulfonamide, mono- or di-C1-C6-alkylsulfonamide, mono- or diarylsulfonamide, mono- or diheteroarylsulfonamide, N-alkyl-N-arylsulfonamide, N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide, urea; mono-di- or tri-substituted urea, wherein the subsitutent(s) is selected from the group consisting of C1-C6-alkyl, aryl, heteroaryl; O-alkylurethane, O-arylurethane, and O-heteroarylurethane;
one of X or Y is hydrogen or an amino-terminal capping group;
the other of X or Y is hydroxy or a carboxy-terminal capping group;
and salts thereof.
As noted above, each xe2x80x9cAxe2x80x9d substituent is selected independently from one another. Consequently, the invention explicitly encompasses both homo-oligomers and polymers, as well as hetero-oligomers and polymers.
Encompassed within the invention are protected forms of the above compounds in which reactive carboxy and amino subtituents are protected by selectively removable (including orthogonally removable) moieties. All substituents used as protecting groups in synthetic organic chemistry are encompassed within the definition. For purposes of this application, a xe2x80x9cprotecting groupxe2x80x9d is a chemical moiety capable of selective addition to and removal from a reactive site to allow manipulation of a chemical entity at sites other than the reactive site. A host of protecting groups are known in the art. An illustrative, non-limiting list of protecting groups includes methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butoxycarbonyl, benzoyl, 4-methylbenzyl, thioanisolyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulfonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxy carbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, and 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl. Expressly included within this definition, without limitation, are carbamate-forming protecting groups such as Boc, Fmoc, Cbz, and the like, and amide-forming protecting groups such as acetyl and the like. Such protecting groups are well known and widely used by those skilled in the art of peptide chemistry.
All stereochemical configurations (single enantiomers, single diastereomers, mixtures thereof, and racemates thereof) of the compounds described above are encompassed within the scope of the invention. In the preferred embodiment, all of the residues share the same absolute configuration (either R or S) about the asymmetric ring carbon in the position a to the exocyclic carbonyl carbon.
The invention is further directed to a method for preparing a combinatorial library of the subject compounds, the method comprising at least two successive iterations of first covalently linking a first {A} subunit via its C terminus to a plurality of separable solid substrates, the first subunit selected from the group recited above for xe2x80x9cA,xe2x80x9d and then randomly dividing the plurality of substrates into at least two sub-groups and deprotecting the first subunits attached to the at least two sub-groups. Then in separate and independent reactions, covalently linking to the first subunit of each of the at least two sub-groups a second subunit independently selected from the above-listed group of {A} residues.
The invention is further drawn to a combinatorial library of oligomers and/or polymers comprising a plurality of different compounds as described above, each compound covalently linked to a solid support, the combinatorial library produced by the process described immediately above.
Another embodiment of the invention is drawn to an array comprising a plurality of compounds as described above at selected, known locations on a substrate or in discrete solutions, wherein each of the compounds is substantially pure within each of the selected known locations and has a composition which is different from other polypeptides disposed at other selected and known locations on the substrate.
The primary advantage and utility of the present invention is that it allows the construction of synthetic peptides having high conformational stability. These synthetic polyamides have practical utility in investigating the biological interactions involving biopolymers, by, for example, constructing support-bound libraries of the compounds and exposing putative pharmacologically active agents to the support-bound compounds. Thus, the compounds and libraries of the compounds can be used to screen other potentially active compounds.
The stable secondary structure of the present compounds also allows them to mimic natural protein secondary structure, thereby allowing targeted disruption of large-molecule interactions (e.g., proteinxe2x80x94protein interactions.) In this instance, the compounds have utility as models of naturally-occuring alpha polypeptides. However, because the present compounds lack alpha peptide linkages, they are less prone to degradation by proteolytic enxymes.
It is also expected that the compounds of the present invention will readily cross biological membranes due to their lower polarity as compared to natural peptides. This is expected based upon the known ability of proline oligomers to cross biological membranes. Because the backbone of the subject oligomers and polymers is linked by tertiary amide bonds, the compounds lack acidic amide protons on the backbone. Additionally, because the compounds are unnatural, they are expected to resist enzymatic cleavage (as noted above). Therefore, the subject oligomers have utility as probes to investigate the ability of non-natural betapeptides to cross biological membranes.
As a natural consequence, the invention is further drawn to the use of these synthetic polyamides as base molecules from which to synthesize large libraries of novel compounds utilizing the techniques of combinatorial chemistry. In addition to varying the primary sequence of the residues, the ring positions of these compounds can be substituted with a wide variety of substituents, such as those described above for R1 and R2. The main advantage here is that substituents placed on the backbone rings do not interfere with the ability of the compounds to adopt a regular secondary structure. Consequently, the subject compounds can be utilized to construct vast libraries having different substituents, but all of which share a stabilized secondary structure. This utility is highly desirable because, as a general principal, chemical structure is responsible for chemical activity. By providing a means for constructing large libraries or arrays of the subject compounds, their structure-activity relationships can be cogently investigated by rational design of libraries or arrays containing systematically altered permutations of the oligomers disclosed herein.
Other aims, objects, and advantages of the invention will appear more fully from a complete reading of the following Detailed Description of the Invention and the attached claims.